Altered glycolysis triggers impaired mitochondrial metabolism and mTORC1 activation in diabetic β-cells

Chronic hyperglycaemia causes a dramatic decrease in mitochondrial metabolism and insulin content in pancreatic β-cells. This underlies the progressive decline in β-cell function in diabetes. However, the molecular mechanisms by which hyperglycaemia produces these effects remain unresolved. Using isolated islets and INS-1 cells, we show here that one or more glycolytic metabolites downstream of phosphofructokinase and upstream of GAPDH mediates the effects of chronic hyperglycemia. This metabolite stimulates marked upregulation of mTORC1 and concomitant downregulation of AMPK. Increased mTORC1 activity causes inhibition of pyruvate dehydrogenase which reduces pyruvate entry into the tricarboxylic acid cycle and partially accounts for the hyperglycaemia-induced reduction in oxidative phosphorylation and insulin secretion. In addition, hyperglycaemia (or diabetes) dramatically inhibits GAPDH activity, thereby impairing glucose metabolism. Our data also reveal that restricting glucose metabolism during hyperglycaemia prevents these changes and thus may be of therapeutic benefit. In summary, we have identified a pathway by which chronic hyperglycaemia reduces β-cell function

Abcc5 Knockout Mice Have Lower Fat Mass and Increased Levels of Circulating GLP-1.

OBJECTIVE: A previous genome-wide association study linked overexpression of an ATP-binding cassette transporter, ABCC5, in humans with a susceptibility to developing type 2 diabetes with age. Specifically, ABCC5 gene overexpression was shown to be strongly associated with increased visceral fat mass and reduced peripheral insulin sensitivity. Currently, the role of ABCC5 in diabetes and obesity is unknown. This study reports the metabolic phenotyping of a global Abcc5 knockout mouse. METHODS: A global Abcc5-/- mouse was generated by CRISPR/Cas9. Fat mass was determined by weekly EchoMRI and fat pads were dissected and weighed at week 18. Glucose homeostasis was ascertained by an oral glucose tolerance test, intraperitoneal glucose tolerance test, and intraperitoneal insulin tolerance test. Energy expenditure and locomotor activity were measured using PhenoMaster cages. Glucagon-like peptide 1 (GLP-1) levels in plasma, primary gut cell cultures, and GLUTag cells were determined by enzyme-linked immunosorbent assay. RESULTS: Abcc5-/- mice had decreased fat mass and increased plasma levels of GLP-1, and they were more insulin sensitive and more active. Recombinant overexpression of ABCC5 protein in GLUTag cells decreased GLP-1 release. CONCLUSIONS: ABCC5 protein expression levels are inversely related to fat mass and appear to play a role in the regulation of GLP-1 secretion from enteroendocrine cells

Stimulus-secretion coupling in L cells and factors affecting secretion of glucagon-like peptide 1

Glucagon-like peptide 1 (GLP-1) is a hormone released from gut endocrine L cells in response to the arrival of nutrients and plays an important role in regulating glucose homeostasis. However, factors and pathways modulating the stimulus-secretion coupling of GLP-1 are not fully understood. Our group previously reported a significant increase in GLP-1 plasma levels from an Abcc5 knockout (Abcc5-/-) mouse model, but the mechanism responsible for this effect remains unknown. Additionally, Abcc5-/- mice generated by other groups have reported an accumulation of various glutamate-conjugated compounds in the brain and other tissues, which could be responsible for driving the altered metabolic phenotype of the Abcc5-/- mice and also potentially affecting their behaviour. In this study, we first characterised the neurobehavioural phenotype of mice lacking the Abcc5 gene. We showed that Abcc5-/- mice displayed a neurobehavioural phenotype consistent with disruption of glutamatergic signalling such as increased startle reactivity, loss of pre-pulse inhibition, and a decreased conditioned fear response. Further electrophysiological evidence implicates that glutamate-gated ion channels, NMDA receptors, might be responsible for the neurobehavioural phenotype of the Abcc5-/- mice. We showed that inhibition of NMDA receptors in an L cell model cell line, GLUTag cells, by various methods resulted in a significant increase in the release of GLP-1. Since native L cells are exposed to different pH conditions alongside the digestive tract, the effect of extracellular pH on GLP-1 secretion was also investigated using GLUTag and primary L cells. Stimulated release of GLP-1 was found to be pH sensitive and an extracellular acidic pH inhibited GLP-1 release in a proton concentration-dependent manner. The mechanism of pH-regulated GLP-1 secretion was explored further to identify the point of proton entry into the cell and possible proton targets within the cell. Importantly, the pH-dependent release of GLP-1 in response to stimulation occurred irrespective of extracellular sodium levels, while chloride and calcium ions prove to be pivotal for GLP-1 secretion. Furthermore, while the production and processing of GLP-1 remained unaffected by an extracellular acidic pH, exocytosis of GLP-1 was inhibited and significantly more secretory granules containing active GLP-1 were shown to be retained intracellularly. Overall, results from this study showed that the activity of peripheral NMDA receptors expressed in L cells and the modulatory effect of extracellular pH plays an important role in the stimulus-secretion coupling of GLP-1 in response to nutrients. These findings provide new target possibilities to stimulate endogenous secretion of GLP-1, a much sought-after therapeutic strategy, for the treatment of obesity and diabetes